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• W2149255022 abstract "Fanconi anemia (FANC) is a heterogeneous genetic disorder characterized by a hypersensitivity to DNA-damaging agents, chromosomal instability, and defective DNA repair. Eight FANC genes have been identified so far, and five of them (FANCA, -C, -E, -F, and -G) assemble in a multinuclear complex and function at least in part in a complex to activate FANCD2 by monoubiquitination. Here we show that FANCA and FANCG are redox-sensitive proteins that are multimerized and/or form a nuclear complex in response to oxidative stress/damage. Both FANCA and FANCG proteins exist as monomers under non-oxidizing conditions, whereas they become multimers following H2O2 treatment. Treatment of cells with oxidizing agent not only triggers the multimeric complex of FANCA and FANCG in vivo but also induces the interaction between FANCA and FANCG. N-Ethylmaleimide treatment abolishes multimerization and interaction of FANCA and FANCG in vitro. Taken together, our results lead us to conclude that FANCA and FANCG uniquely respond to oxidative damage by forming complex(es) via intermolecular disulfide linkage(s), which may be crucial in forming such complexes and in determining their function. Fanconi anemia (FANC) is a heterogeneous genetic disorder characterized by a hypersensitivity to DNA-damaging agents, chromosomal instability, and defective DNA repair. Eight FANC genes have been identified so far, and five of them (FANCA, -C, -E, -F, and -G) assemble in a multinuclear complex and function at least in part in a complex to activate FANCD2 by monoubiquitination. Here we show that FANCA and FANCG are redox-sensitive proteins that are multimerized and/or form a nuclear complex in response to oxidative stress/damage. Both FANCA and FANCG proteins exist as monomers under non-oxidizing conditions, whereas they become multimers following H2O2 treatment. Treatment of cells with oxidizing agent not only triggers the multimeric complex of FANCA and FANCG in vivo but also induces the interaction between FANCA and FANCG. N-Ethylmaleimide treatment abolishes multimerization and interaction of FANCA and FANCG in vitro. Taken together, our results lead us to conclude that FANCA and FANCG uniquely respond to oxidative damage by forming complex(es) via intermolecular disulfide linkage(s), which may be crucial in forming such complexes and in determining their function. Fanconi anemia (FANC) 1The abbreviations used are: FANC, Fanconi anemia; MMC, mitomycin C; DTT, dithiothreitol; GST, glutathione S-transferase; E3, ubiquitin-protein isopeptide ligase.1The abbreviations used are: FANC, Fanconi anemia; MMC, mitomycin C; DTT, dithiothreitol; GST, glutathione S-transferase; E3, ubiquitin-protein isopeptide ligase. is an autosomal recessive disorder characterized by chromosomal instability and defective DNA repair, and FANC-deficient cells exhibit extreme sensitivity toward oxygen and DNA-cross-linking agents such as diepoxybutane and mitomycin C (1Joenje H. Oostra A.B. Wijker M. di Summa F.M. van Berkel C.G. Rooimans M.A. Ebell W. van Weel M. Pronk J.C. Buchwald M. Arwert F. Am. J. Hum. Genet. 1997; 61: 940-944Abstract Full Text PDF PubMed Scopus (252) Google Scholar, 2Joenje H. Patel K.J. Nat. Genet. 2001; 2: 446-453Crossref Scopus (502) Google Scholar, 3Howlett N.G. Taniguchi T. Olson S. Cox B. Waisfisz Q. De Die-Smulders C. Persky N. Grompe M. Joenje H. Pals G. Ikeda H. Fox E.A. D'Andrea A.D. Science. 2002; 297: 534-537Crossref PubMed Scopus (955) Google Scholar). The gene products of eight complementation groups of FANC have been identified and cloned (FANCA, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, and FANCL) (1Joenje H. Oostra A.B. Wijker M. di Summa F.M. van Berkel C.G. Rooimans M.A. Ebell W. van Weel M. Pronk J.C. Buchwald M. Arwert F. Am. J. Hum. Genet. 1997; 61: 940-944Abstract Full Text PDF PubMed Scopus (252) Google Scholar, 2Joenje H. Patel K.J. Nat. Genet. 2001; 2: 446-453Crossref Scopus (502) Google Scholar, 3Howlett N.G. Taniguchi T. Olson S. Cox B. Waisfisz Q. De Die-Smulders C. Persky N. Grompe M. Joenje H. Pals G. Ikeda H. Fox E.A. D'Andrea A.D. Science. 2002; 297: 534-537Crossref PubMed Scopus (955) Google Scholar). Mutations in any of the eight different genes lead to FANC disease, a degree of genetic heterogeneity comparable with that of other DNA repair disorders, suggesting that each group represents a distinct protein.FANCA and FANCG proteins are part of a large nuclear protein complex required for their function, and the disruption of this complex results in the specific cellular and clinical phenotype common to most FANC complementation groups (4Garcia-Higuera I. Kuang Y. Naf D. Wasik J. D'Andrea A.D. Mol. Cell. Biol. 1999; 19: 4866-4873Crossref PubMed Scopus (199) Google Scholar). FANCA gene encodes a 162-kDa phosphoprotein and its phosphorylation correlated with FANCA/FANCC protein accumulation in the nucleus (5Yamashita T. Kupfer G.M. Naf D. Suliman A. Joenje H. Asano S. D'Andrea A.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13085-13090Crossref PubMed Scopus (107) Google Scholar). FANCA mutant cells isolated from a FANC patient were defective in their phosphorylation and failed to bind to FANCC. Furthermore, a mutant FANCA protein failed to complement the mitomycin C (MMC) sensitivity of FANCA–/– cells, suggesting that FANCA phosphorylation may be involved in FANCC interaction, nuclear localization of FANCA, or its function in cross-link repair. FANCG gene encodes a 65-kDa protein that has been identified as human XRCC9. XRCC9 (FANCG) complements the Chinese hamster ovary mutant UV-40 cell line that is hypersensitive to UV, ionizing radiation, simple alkylating agents, and DNA-cross-linking agents (6Busch D.B. Zdzienicka M.Z. Natarajan A.T. Jones N.J. Overkamp W.J. Collins A. Mitchell D.L. Stefanini M. Botta E. Albert R.B. Liu N. White D.A. van Gool A.J. Thompson L.H. Mutat. Res. 1996; 363: 209-221Crossref PubMed Scopus (28) Google Scholar, 7Liu N. Lamerdin J.E. Tucker J.D. Zhou Z.Q. Walter C.A. Albala J.S. Busch D.B. Thompson L.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9232-9237Crossref PubMed Scopus (69) Google Scholar). The mutant cells also show a high level of spontaneous chromosomal aberrations that can be fully corrected by introduction of XRCC9 cDNA transformants (7Liu N. Lamerdin J.E. Tucker J.D. Zhou Z.Q. Walter C.A. Albala J.S. Busch D.B. Thompson L.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9232-9237Crossref PubMed Scopus (69) Google Scholar). The possibility of the involvement of FANC proteins in DNA repair was strengthened by recent findings on the interaction of FANCD1 with BRCA1 following DNA damage (8Garcia-Higuera I. Taniguchi T. Ganesan S. Meyn M.S. Timmers C. Hejna J. Grompe M. D'Andrea A.D. Mol. Cell. 2001; 7: 249-262Abstract Full Text Full Text PDF PubMed Scopus (1019) Google Scholar). FANCD1 is identical to BRCA2 gene and is unique among FANC genes in that it is essential for the formation of Rad51 foci in response to ionizing radiation (9Godthelp B.C. Artwert F. Joenje H. Zdzienicka M.Z. Oncogene. 2002; 21: 5002-5005Crossref PubMed Scopus (88) Google Scholar), suggesting that it may be involved in homologous recombination-mediated strand break repair.Cells lacking FANC gene showed a hypersensitive phenotype following H2O2 treatment, suggesting a role for FANC proteins in redox signaling and repair of oxidative DNA damages (Refs. 10Cumming R.C. Lightfoot J. Beard K. Youssoufian H. O'Brien P.J. Buchwald M. Nat. Med. 2001; 7: 814-820Crossref PubMed Scopus (205) Google Scholar, 11Futaki M. Igarashi T. Watanabe S. Kajigaya S. Tatsuguchi A. Wang J. Liu J.M. Carcinogenesis. 2002; 23: 67-72Crossref PubMed Scopus (89) Google Scholar, 12Zunino A. Degan P. Vigo T. Abbondandolo A. Mutagenesis. 2001; 16: 283-288Crossref PubMed Scopus (33) Google Scholar, 13Saadatzadeh M.R. Bijangi-Vishehsaraei K. Hong P. Bergmann H. Haneline L.S. J. Biol. Chem. 2004; 279: 16805-16812Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar and data not shown). Interaction between FANCA and FANCG was well established by coimmunoprecipitation, cellular localization, and yeast two-hybrid analysis (4Garcia-Higuera I. Kuang Y. Naf D. Wasik J. D'Andrea A.D. Mol. Cell. Biol. 1999; 19: 4866-4873Crossref PubMed Scopus (199) Google Scholar, 14Waisfisz Q. de Winter J.P. Kruyt F.A. de Groot J. van der Weel L. Dijkmans L.M. Zhi Y. Arwert F. Scheper R.J. Youssoufian H. Hoatlin M.E. Joenje H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10320-10325Crossref PubMed Scopus (124) Google Scholar, 15Kruyt F.A. Abou-Zahr F. Mok H. Youssoufian H. J. Biol. Chem. 1999; 274: 34212-34218Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 16Reuter T. Herterich S. Bernhard O. Hoehn H. Gross H.J. Blood. 2000; 95: 719-720Crossref PubMed Google Scholar, 17Huber P.A. Medhurst A.L. Youssoufian H. Mathew C.G. Biochem. Biophys. Res. Commun. 2000; 268: 73-77Crossref PubMed Scopus (24) Google Scholar, 18Gordon S.M. Buchwald M. Blood. 2003; 102: 136-141Crossref PubMed Scopus (52) Google Scholar). Although the detailed functions of FANC proteins have yet to be determined, there is a growing consensus on the role for FANC proteins in DNA repair and damage signaling pathways. In this study, we investigated the interaction of key FANC proteins following oxidative damage(s). We found that FANCA and FANCG are multimerized and/or form a complex via intermolecular disulfide linkage(s) in response to oxidative damage. Nuclear localization as well as FANCA-FANCG interaction occurs concomitantly with monoubiquitination of FANCD2 upon H2O2 treatment, suggesting that FANCA-FANCG interaction may be an early response to oxidative damage that plays a crucial role in DNA repair and damage signaling pathways.MATERIALS AND METHODSAntibodies and Chemicals—Anti-FANCA and -FANCG antisera (polyclonal) were generated in rabbits using immunogenic peptides with FANCA residues (525–544 (ENMGLYEDLSSAGDITEPHS) and 1230–1249 (HFAIQQVREENIRKQLKKLD)) and FANCG residues (101–120 (ERVLETQEQQGPRLEQGLRE) and 604–622 (DRDAFLEEFRTSLPKSCDL)). Anti-FLAG and anti-Myc antibodies were obtained from Sigma, and anti-FANCD2 antibody (monoclonal antibody NB100-316) was from Novus Biologicals. N-Ethylmaleimide, β-mercaptoethanol, and MMC were from Sigma, and DTT was from Roche Applied Science. H2O2 was obtained from Fisher.Cloning of FANCA and FANCG Genes—The cDNA for human FANCG was subcloned into pcDNA6.2-DEST (Invitrogen) by PCR using the forward primer 5′-CACCACCATGGAGCAGAAGCTTATTTCGGAGGAAGACCTATCC CGCCAGACCACCTCTGTGGGC-3′ and the reverse primer 5′-TCACAGGTCACAAGACTTTGGCAG-3′. Both primers contain a CACC sequence for recombination into pcDNA6.2-DEST, and the forward primer also encodes a Myc tag. Positive clones were isolated and confirmed by DNA sequencing. Human FANCA cDNA was subcloned into pFLAG (Kodak) at NotI and HindIII by PCR using the forward primer 5′-TCTGCGGCCCGATGTCCGACTCGTGGGTCCCG-3′ and reverse primer 5′-TCTAAGCTTTCAGAAGAGATGAGGCTCCTG-3′. Positive clones were confirmed by restriction enzyme analysis and DNA sequencing. Positive clones (plasmids) were transfected into COS cells for expression. For GST-FANCG expression, human FANCG was subcloned into pAcG2T (Pharmingen) at EcoRI by PCR using the forward primer 5′-CGGGAATTCATTCCCGCCAGACCACC-3′ and the reverse primer 5′-CGGGAATTCCTACAGGTCACAAGAC-3′. Positive clones were confirmed by restriction analysis and DNA sequencing.Cell Cultures and Preparation of Cell Extracts—COS cells and HeLa cells were grown in Dulbecco's modified Eagle's medium and Dulbecco's modified Eagle's medium/F-12 (Invitrogen), respectively, which were supplemented with 10% fetal calf serum (Invitrogen), penicillin (10 units/ml, Sigma), and streptomycin (0.1 mg/ml, Sigma). For all experiments in this study, cells were in exponential growth phase. Cells were treated with H2O2 or MMC for 2 h at the indicated concentrations at 37 °C. After reagent exposure, cells were washed with phosphate-buffered saline and lysed directly on the 100-mm plates by adding 500 μl of cold lysis buffer (1% Nonidet P-40 in phosphate-buffered saline in the presence of 1 mm Na3VO4, 1 mm NaF, and mammalian protease inhibitor mixture) to each and scraping rapidly with a cell scraper. The cell lysates were centrifuged for 30 min at 20,000 × g, and the supernatant (the Nonidet P-40-soluble protein fraction) was harvested. The pellet (the Nonidet P-40-insoluble protein fraction) was washed once with lysis buffer and dissolved with SDS-sample loading buffer. These soluble and insoluble proteins were immediately analyzed by electrophoresis/immunoblotting or immunoprecipitation.Protein Purification—Anti-FLAG M2-agarose was used in purifying FLAG-FANCA protein according to the manufacturer's instructions (Sigma). The supernatants from transfected COS cell lysates were incubated at 4 °C for at least 4 h with anti-FLAG M2 affinity gel (Sigma) that had been preequilibrated in phosphate-buffered saline buffer. The beads were then washed three times with 1% Nonidet P-40 in phosphate-buffered saline buffer. In the final step, the M2 column was eluted with 3 column volumes of 500 μg/ml FLAG peptide. GST-FANCG proteins were expressed in Sf9 insect cells following recombinant baculovirus infection (19Stigger E. Dean F.B. Hurwitz J. Lee S.-H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 579-583Crossref PubMed Scopus (58) Google Scholar) and purified by glutathione-Sepharose resin (Amersham Biosciences) and by fast protein liquid chromatography on a 1-ml HiTrap Q column (Amersham Biosciences) (20You J.S. Wang M. Lee S.H. J. Biol. Chem. 2003; 278: 7476-7485Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar).SDS-PAGE and Western Blot Analysis—Cell lysates (25 μg/lane) or purified proteins were resolved on 6–8% SDS-polyacrylamide gels under reducing conditions (10 mm DTT or 1% β-mercaptoethanol) or under non-reducing conditions. Proteins were then transferred to a polyvinylidene difluoride membrane and probed with an anti-FANCA or anti-FANCG antibody (a rabbit polyclonal IgG) or with an anti-FANCD2 antibody (monoclonal mouse IgG, Novus Biologicals) followed by horseradish peroxidase-conjugated secondary antibody. Proteins were visualized by using the ECL system (Amersham Biosciences).Immunoprecipitation—Cell lysates were clarified by centrifugation in a microcentrifuge for 10 min at 8,000 × g at 4 °C. The supernatants were removed and incubated with anti-FLAG M2 antibody for 2 h at 4 °C. The mixture was then added to a 60-μl packed gel volume of protein G-agarose affinity beads (prewashed and equilibrated in lysis buffer) and incubated for 1 h with mixing at 4 °C. The beads were collected by centrifugation for 2 min at 2,000 × g, and the supernatants were removed by aspiration. The pellets were washed three times with 1 ml of lysis buffer/wash and collected by centrifugation as described. After washing, the affinity-bead pellets were each suspended in the presence of 30 μl of 2× Laemmli sample buffer (21Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205998) Google Scholar) and analyzed by SDS-PAGE and immunoblotting.RESULTSMultimerization of FANC Proteins under Oxidizing Conditions—Increasing evidence points to a role for FANC proteins in redox signaling and the repair of oxidative damages (22Demple B. Harrison L. Annu. Rev. Biochem. 1994; 63: 915-948Crossref PubMed Scopus (1286) Google Scholar, 23Kelley M.R. Tritt R. Xu Y. New S. Freie B. Clapp D.W. Deutsch W.A. Mutat. Res. 2001; 485: 107-119Crossref PubMed Scopus (18) Google Scholar, 24Pagano G. Youssoufian H. BioEssays. 2003; 25: 589-595Crossref PubMed Scopus (57) Google Scholar). In accordance with these findings, cells lacking FANC genes showed hypersensitivity to H2O2 treatment (Ref. 13Saadatzadeh M.R. Bijangi-Vishehsaraei K. Hong P. Bergmann H. Haneline L.S. J. Biol. Chem. 2004; 279: 16805-16812Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar and data not shown). Because several FANC proteins (FANCA, FANCC, and FANCG) contain unusually high numbers of cysteines, we wondered whether some of these residues were directly involved in redox regulation. To examine possible redox regulation of FANC proteins, insect cells were transfected with recombinant baculovirus expressing GST-FANCG. Purified FANCG protein was analyzed by SDS-PAGE following a brief air oxidation and the addition of reducing agent. Purified GST-FANCG migrated as multimers (>250 kDa in size) under oxidizing conditions, whereas it migrated as a monomer with increasing amounts of DTT (Fig. 1A). The transition from monomer to multimer likely occurred through the formation of intermolecular disulfide linkage(s) on FANCG but not on the GST portion of the fusion protein because purified GST did not form multimers regardless of its redox status (Fig. 1B). It is interesting to note that GST-FANCG (90 kDa) migrated more than one form of multimers in a range from 180 kDa to a much larger size under oxidizing conditions (Fig. 1, A and C).Redox-mediated Transition of Monomers/Multimers of FANCA and FANCG Expressed in COS Cells—We next examined whether a redox potential affects monomeric/multimeric change of FANC proteins. COS cells either were transfected with a mammalian vector encoding FANCA or FANCG or were cotransfected with both FANCA and FANCG vectors. Cell lysates were examined for expression of FANCA and FANCG by Western blotting. A stable expression of FANCA was observed regardless of the presence of FANCG (Fig. 2A, lanes 1 and 2). On the other hand, FANCG protein was stably expressed only in the presence of FANCA and was hardly detected without FANCA (Fig. 2A, lane 3 versus lane 4), suggesting that FANCA is necessary for stable expression of FANCG in mammalian cells (25Garcia-Higuera I. Kuang Y. Denham J. D'Andrea A.D. Blood. 2000; 96: 3224-3230Crossref PubMed Google Scholar). Similar to an in vitro study with purified GST-FANCG (Fig. 1A), FANCA multimers were converted to a monomer following incubation with increasing amounts of DTT (Fig. 2B), suggesting that multimerization of FANCA occurs through intermolecular disulfide linkage(s). FANCA and FANCG in COS cells exist as monomers under reduced conditions (Fig. 2C, lane 1) but are converted to various sizes of multimer(s) under non-reducing conditions (Fig. 2C, lane 2).Fig. 2Multimerization of FANCA and FANCG expressed in COS cells under non-reducing conditions. A, COS cells were transfected with mammalian vector (pcDNA) expressing FLAG-FANCA (lane 1), Myc-FANCG (lane 3), or both (lanes 2 and 4). Cell extracts (60 μg) were prepared from each group of transfected cells, loaded onto SDS-polyacrylamide gels, and analyzed by Western blotting using either an anti-FANCA (lanes 1 and 2) or anti-FANCG (lanes 3 and 4) antibody. B, transition of FANCA multimers into a monomer in the presence of DTT. Cell extracts from COS cells transfected with FANCA-expressing vector were oxidized on air overnight prior to the addition of increasing amounts of DTT. Samples were then analyzed by SDS-PAGE and Western blotting using an anti-FANCA antibody. C, transition of multimers of FANCA/FANCG proteins into a monomer in the presence of DTT. Cell extracts were prepared from COS cells cotransfected with FANCA and FANCG. Extracts were incubated at 4 °C overnight either in the presence (lane 1) or absence (lane 2) of 10 mm DTT prior to loading onto SDS-polyacrylamide gels and Western analysis. Ku70 was used as an internal control for FANCA and FANCG in cell extracts.View Large Image Figure ViewerDownload (PPT)Treatment of Cells with Mitomycin C Also Induces a Multimeric Complex of FANCA and FANCG in Vivo—FANC cells are highly sensitive to MMC, a DNA-damaging agent that primarily induces interstrand cross-link DNA damage but also can cause oxidative damage as well as other types of DNA damages (intrastrand cross-link and monoadduct) (24Pagano G. Youssoufian H. BioEssays. 2003; 25: 589-595Crossref PubMed Scopus (57) Google Scholar, 26D'Andrea A.D. Grompe M. Nat. Rev. Cancer. 2003; 3: 23-34Crossref PubMed Scopus (665) Google Scholar). We therefore examined whether MMC treatment also induces a multimeric complex of FANC proteins in vivo. COS cells expressing both FANCA and FANCG were treated for 2 h with either H2O2 or MMC and were examined for multimerization of FANC proteins. Similar to H2O2 treatment, cells treated with MMC induced multimerization of FANCA (Fig. 3A, top). FANCA multimers were converted back to monomers when the samples were subjected to SDS-PAGE in the presence of DTT (Fig. 3A, middle). Concomitant analysis of FANCG also showed multimerization of FANCG in response to MMC treatment (Fig. 3B), suggesting that multimerization of both FANCA and FANCG is induced by MMC.Fig. 3COS cells treated with either H2O2 or a DNA-damaging agent (MMC) induced a multimeric complex of FANCA and FANCG in vivo. COS cells cotransfected with FLAG-FANCA- and Myc-FANCG-expressing vectors were treated with an increasing amount of either H2O2 or MMC for 2 h. After harvesting the cells, extracts were prepared and analyzed for multimerization of FANCA (A) or FANCG (B) by SDS-PAGE. Where indicated (A and B, middle panel), 10 mm DTT was added before subjecting the extracts to SDS-PAGE. Ku70 in the bottom panel (A and B) was used as an internal control.View Large Image Figure ViewerDownload (PPT)FANCA-FANCG Interaction Is Significantly Enhanced in Response to H2O2 or MMC—Because redox potential affects the multimerization of both FANCA and FANCG, we examined whether it also influences FANCA-FANCG interaction. A stable interaction between FANCA and FANCG was observed in COS cells transfected with FLAG-FANCA and Myc-FANCG (Fig. 4A). Compared with non-treated control cells, COS cells treated with H2O2 induced multimerization of FANCA (Fig. 4B, top, lanes 2–4 versus lanes 5–7) as well as interaction between FANCA and FANCG; the latter was evidenced by a significantly higher amount of FANCG coprecipitated with FANCA in a FLAG-antibody pulldown assay (Fig. 4B, bottom, lanes 2–4 versus lanes 5–7).Fig. 4Interaction between FANCA and FANCG in vivo was significantly stimulated by H2O2 treatment. A, cell extracts (500 μg) prepared from COS cells cotransfected with FLAG-FANCA- and Myc-FANCG-expressing vectors were incubated with an anti-FLAG antibody for 2 h for immunoprecipitation using protein A-Sepharose beads. Proteins bound to the beads were eluted with FLAG peptide and analyzed in the presence of 10 mm DTT for FANCA and FANCG by SDS-PAGE and silver staining (lane 1) and Western blotting using anti-FANCA and anti-FANCG antibodies (lane 2). M represents the lane with protein markers. B, COS cells transfected with FLAG-FANCA- and Myc-FANCG-expressing vectors were either mock-treated (lanes 1–4) or treated with 1 mm H2O2 (lanes 5–7) for 2 h. Cell extracts (50 μg) were incubated with an anti-FLAG antibody (Flag Ab) for immunoprecipitation. Total immunoprecipitates (lanes 2 and 5) were further eluted with FLAG peptide to separate the fraction bound to the beads (lanes 3 and 6) and the eluant (lanes 4 and 7). T, B, and E stand for total immunoprecipitates, fraction bound to the beads, and eluant/unbound fraction, respectively. Samples were analyzed by SDS-PAGE and Western blotting using an anti-FANCA (top panel) or anti-FANCG (bottom panel) antibody. In the bottom panel, DTT (10 mm) was added to the sample prior to SDS-PAGE to fairly quantify Myc-FANCG.View Large Image Figure ViewerDownload (PPT)Because MMC treatment of cells induced multimerization of FANC proteins (Fig. 3), we also examined whether MMC treatment affected the FANCA-FANCG interaction (Fig. 5). COS cells expressing FLAG-FANCA and Myc-FANCG were treated with either H2O2 or MMC and were examined for coprecipitation of FANCG following immunoprecipitation of FLAG-FANCA using an anti-FLAG antibody (Fig. 5). Association of Myc-FANCG with FLAG-FANCA was significantly increased following treatment of cells with either H2O2 or MMC (Fig. 5A, lane 4 versus lanes 5–6). Most, if not all, of the FANCG coprecipitated with FANCA was in a multimerized form of FANCG that migrated at >250 kDa in size in the absence of DTT (Fig. 5B, lanes 5 and 6). We also observed that N-ethylmaleimide treatment abolishes multimerization and interaction of FANCA and FANCG in vitro (data not shown). Together, our results suggest that the FANCA-FANCG interaction may occur through intermolecular disulfide linkage(s) in a oxidative damage-dependent manner.Fig. 5Interaction between FANCA and FANCG is induced in vivo upon treatment with either H2O2 or MMC. COS cells were cotransfected with expression vectors for FLAG-FANCA and Myc-FANCG, and the cells were untreated (A–C, lanes 3 and 4), treated with 1 mm H2O2 (A–C, lane 5), or treated with 1 μm MMC (A–C, lane 6) for 2 h. Cell extracts (200 μg) were incubated with an anti-FLAG antibody (Flag-Ab) for 2 h prior to addition of protein A-Sepharose beads for immunoprecipitation. Lanes 1 and 2 (A–C) contained no cell extracts. Following immunoprecipitation, the samples were analyzed by SDS-PAGE and Western blotting using an anti-FANCG antibody (A and B) or an anti-FANCA antibody (C). For analysis, samples were treated with 10 mm DTT (A) or 0 mm DTT (B and C) for 1 h just before being subjected to SDS-PAGE. In B, ** represents an unidentified band that did not match the monomer form of Myc-FANCG.View Large Image Figure ViewerDownload (PPT)DISCUSSIONFANC cells exhibit a defect in damage-induced cell cycle arrest, a high level of chromosomal aberrations, and hypersensitivity to DNA-cross-linking agents. FANC mutant cells from groups A, B, C, E, F, and G were defective in forming the FANC protein complex (4Garcia-Higuera I. Kuang Y. Naf D. Wasik J. D'Andrea A.D. Mol. Cell. Biol. 1999; 19: 4866-4873Crossref PubMed Scopus (199) Google Scholar, 14Waisfisz Q. de Winter J.P. Kruyt F.A. de Groot J. van der Weel L. Dijkmans L.M. Zhi Y. Arwert F. Scheper R.J. Youssoufian H. Hoatlin M.E. Joenje H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10320-10325Crossref PubMed Scopus (124) Google Scholar, 27Naf D. Kupfer G.M. Suliman A. Lambert K. D'Andrea A.D. Mol. Cell. Biol. 1998; 18: 5952-5960Crossref PubMed Scopus (107) Google Scholar), and FANCG (XRCC9) protein is required for binding of the FANCA and FANCC proteins (4Garcia-Higuera I. Kuang Y. Naf D. Wasik J. D'Andrea A.D. Mol. Cell. Biol. 1999; 19: 4866-4873Crossref PubMed Scopus (199) Google Scholar, 14Waisfisz Q. de Winter J.P. Kruyt F.A. de Groot J. van der Weel L. Dijkmans L.M. Zhi Y. Arwert F. Scheper R.J. Youssoufian H. Hoatlin M.E. Joenje H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10320-10325Crossref PubMed Scopus (124) Google Scholar), suggesting that FANC proteins form a large multiprotein complex that may be necessary for their function in damage signaling and repair. In this study we examined the interaction of FANC proteins in response to oxidative stress. Our study showed that oxidative DNA damage(s) induced multimerization and interaction of FANC proteins that concomitantly occurred with monoubiquitination of FANCD2.Most redox reactions occur at the thiol group of cysteine, the prominent redox-active amino acid in proteins. FANCA and FANCG possess unusually high numbers of cysteines (38 and 16, respectively), although they do not have any cysteine-mediated motifs such as zinc finger, ring finger, etc. All three FANC proteins formed various multimers under oxidizing conditions in both native and denaturing PAGE, suggesting that the formation of the FANC complex is induced upon redox change via intermolecular disulfide linkage(s) between FANC proteins. Many transcription factors and DNA-binding proteins harboring zinc finger motifs are regulated by redox-mediated change at intramolecular disulfide linkage(s) (28Berg J.M. Shi Y. Science. 1996; 271: 1081-1085Crossref PubMed Scopus (1659) Google Scholar, 29Park J.S. Wang M. Park S.J. Lee S.H. J. Biol. Chem. 1999; 274: 29075-29080Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). On the other hand, the control of protein function through the oxidation state of conserved cysteines is also attributed to intermolecular disulfide linkage(s). Glutathione forms a dimer via disulfide linkage upon redox change and is an important cellular defense against oxidative stress (30Thomas J.A. Poland B. Honzatko R. Arch. Biochem. Biophys. 1995; 319: 1-9Crossref PubMed Scopus (363) Google Scholar). Fos/Jun (31Abate C. Patel L. Rauscher III, F.J. Curran T. Science. 1990; 249: 1157-1161Crossref PubMed Scopus (1373) Google Scholar, 32Okuno H. Akahori A. Sato H. Xanthoudakis S. Curran T. Iba H. Oncogene. 1993; 8: 695-701PubMed Google Scholar) and NF-Y complex (33Nakshatri H. Bhat-Nakshatri P. Currie R.A. J. Biol. Chem. 1996; 271: 28784-28791Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) are the transcription factors that form heterodimers for their function in response to oxidative stress. It should be pointed out, however, that some of the complexes involving disulfide linkage(s), such as gp70-Pr15E interaction, may not be regulated by redox change because the complexes are resistant to treatment with reducing agent(s) (34Opstelten D.J. Wallin M. Garoff H. J. Virol. 1998; 72: 6537-6545Crossref PubMed Google Scholar). Given the fact that redox change facilitates physical interaction of FANCA with FANCG, the FANCA-FANCG interaction is likely controlled by intermolecular disulfide linkage(s). Identification of cysteine sites on FANCA and FANCG would be necessary to clarify the roles of the FANCA-FANCG complex in damage signaling and DNA repair" @default.
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• W2149255022 title "Oxidative Stress/Damage Induces Multimerization and Interaction of Fanconi Anemia Proteins" @default.
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